Project Summary More than 250 million people are chronically infected with hepatitis B virus (HBV), living at increased risk of liver failure, cancer, and early death. Though there is a vaccine to prevent HBV infection, it does not prevent vertical transfer, nor does it help the millions of people already infected. While there are therapies (e.g. nucleoside analogs) to suppress virus replication, treatment is life-long and rarely leads to cure. Achieving a functional cure for chronic HBV infection therefore represents a major global unmet medical need. Fortunately, new classes of HBV inhibitors are being developed and some have already entered clinical trials, but many fundamental (and clinically-relevant) aspects of HBV biology remain unanswered. The broad long-term objectives of this project are to address knowledge gaps in the field using a new cell culture-based method we developed to initiate HBV replication with RNA. Specifically, we capitalize on the most unique features of this technology to (i) address a practical problem of drug development?antiviral resistance, (ii) to fill gaps in our fundamental knowledge of HBV protein translation and genome replication, and (iii) as a discovery tool to identify host factors that restrict or promote the HBV lifecycle. There is currently no way to assess antiviral resistance with existing HBV cell culture-based systems. With our RNA approach, we take advantage of the fact that phage polymerases (e.g. T7) commonly used to in vitro-transcribe RNA are error-prone. As such, initiating HBV replication with RNA rather than DNA (like most systems) allows a diverse population of viral variants to be sampled. As we show with compelling preliminary data, this sequence diversity coupled with deep sequencing technology makes it possible to select for and detect rare HBV drug-resistant viral variants. Aim 1 of this proposal is to further develop this technology. Initiating HBV replication with RNA largely eliminates the background signal that contaminates HBV qPCR reactions, and since not all viral RNAs are required to initiate replication, some viral proteins are made only if the viral lifecycle progresses and the viral DNA template?covalently closed circular DNA (cccDNA)?is established. We capitalize on this in Aim 2 to study fundamental aspects of HBV protein translation and genome replication, and in Aim 3 to discover virus-host interactions. Specifically we use the method in Aim 2 to study mutations that frequently arise in chronically infected individuals and differences in HBV genotypes. We use the method in Aim 3 as the basis for a genome-wide CRISPR knockout screen to identify HBV host factors. Until we have a solution for eliminating chronic HBV, continuing and new fundamental studies of HBV biology are needed to identify new strategies that can be explored for therapeutic intervention. The new RNA- based system we developed to study HBV biology and assess antiviral resistance align well with the NIH's strategic plan to cure chronic HBV and the work we propose will contribute significantly to this ongoing effort.